洗毛用枯草芽孢杆菌产蛋白酶发酵条件优化

Optimization of Protease Production Fermentation Conditions by Bacillus subtilis

用响应面法(Response Surface Methodology,RSM)对枯草芽孢杆菌(Bacillus subtilis)液体发酵产蛋白酶的发酵条件进行快速优化。首先运用逐因子试验确定Bacillus subtilis产蛋白酶的最佳碳源和氮源分别为糊精和玉米粉。在此基础上,通过Plackett-Burman设计对影响其产酶相关因素进行评估,并筛选出具有显著效应的3个因素:糊精、玉米粉和酵母膏。在用最陡爬坡试验逼近以上3个因子的最大响应区域后,采用RSM法对以上3个显著因子的最佳水平范围进行研究,通过对二次多项回归方程求解,确定其最优发酵条件为:糊精11.568g/L,玉米粉22.462g/L,酵母膏10.202g/L,NaCl 5g/L。初始pH值为7.0,37℃培养48h。最终优化后的酶活达到3281.79U/mL,比初始酶活2463.95U/mL提高了33.19%,证明RSM法优化洗毛用Bacillus subtilis产蛋白酶发酵工艺是可行的。

The response surface methodology （RSM） was used to optimize the immersion fermentation conditions for protease production by Bacillus subtilis. Firstly, dextrin and cornmeal were screened out as the most suitable carbon and nitrogen sources by using single factor experiment. Based on these, the assay Plackett-Burman was designed to evaluate relative factors related to protease production and three statistically significant factors （dextrin, cornmeal and yeast extract） were selected. And then the steepest ascent method was applied to define the optimal response region for these three factors, the RSM was used to optimize the above critical factors, and to find out the optimal concentration levels and the relationships between these factors. By solving the quadratic regression model equation by using appropriate statistic methods, the optimal fermentation concentrations were determined, with dextrin of 11.568g/L, cornmeal of 22.462g/L, yeast extract of 10.202g/L, NaCl of 5g/L, initial pH of 7.0, and 37℃ incubation for 48h. Protease activity after optimization was increased from 2463.95U/mL to 3281.79U/mL, an increase by 33.19%. The experiment data under various conditions have validated the theoretical values.